Normal development, growth, and homeostasis in multicellular organisms require a careful balance between the production and destruction of cells in tissues throughout the body. Cell division is a carefully coordinated process with numerous checkpoints and control mechanisms. These mechanisms are designed to regulate DNA replication and to prevent inappropriate or excessive proliferation. In contrast, programmed cell death is the genetically controlled process by which unneeded or damaged cells can be eliminated without causing the tissue destruction and inflammatory responses that are often associated with acute injury and necrosis.
The term "apoptosis" was first used by Kerr, J. F. et al. (1972; Br. J. Cancer 26:239-257) to describe the morphological changes that characterize cells undergoing programmed cell death. Apoptotic cells have a shrunken appearance with an altered membrane lipid content and highly condensed nuclei. Apoptotic cells are rapidly phagocytosed by neighboring cells or macrophages without leaking their potentially damaging contents into the surrounding tissue.
The processes and mechanisms regulating apoptosis are highly conserved throughout the phylogenetic tree. Indeed, much of our current knowledge about apoptosis is derived from studies of the nematode, Caenorhabditis elegans and the fruit fly, Drosophila melanogaster (See for example, Steller, H. (1995) Science 267:1445-1449, and references therein). Dysregulation of apoptosis has recently been recognized as a significant factor in the pathogenesis of human disease. For example, inappropriate cell survival can cause or contribute to many diseases such as cancer, autoimmune diseases, and inflammatory diseases. In contrast, increased apoptosis can cause immunodeficiency diseases such as AIDS, neurodegenerative disorders, and myelodysplastic syndromes (reviewed by Thompson, C. B. (1995) Science 267:1456-1462).
A variety of ligands and their cellular receptors, enzymes, tumor suppressors, viral gene products, pharmacological agents, and inorganic ions have important positive or negative roles in regulating and implementing the apoptotic destruction of a cell (Steller, H., supra; Thompson, C. B., supra). Although many different extra- and intracellular signals can trigger apoptosis (cf. Raff, M. C. (1992) Nature 356:397-400, Raff, M. C. et al. (1993) Science 262:695-700, and Steller, H. and M. E. Grether, (1994) Neuron 13:1269-1274), these signals probably all converge on a common mechanism that ultimately causes the cell to die.
The mouse apoptosis-linked gene 2 (ALG-2) is one of six clones that has been identified using an in vitro "death trap" model for apoptosis. In this model, 3DO hybridoma cells are transformed with cloned cDNAs and then induced to undergo programmed cell death by cross-linking their T cell receptors. Certain cDNAs afford protection to the transformed 3DO cells either by expressing a protein which inhibits apoptosis or by expressing an antisense RNA which blocks the synthesis of a required protein (Vito, P. et al. (1996) Science 271:521-525).
Analysis of the recovered ALG-2 clone indicates that it is of the latter type, thereby identifying ALG-2 as a protein that is essential for programmed cell death. Northern blot analyses detect a single, .about.1.3 KB ALG-2 transcript that is constitutively expressed in all adult mouse tissues; expression is highest in the thymus and liver and lowest in the testes and skeletal muscles. Constitutive expression in normal tissue implies that ALG-2 is probably in an inactive state until the apoptosis pathway is triggered. Apoptosis induced by T cell receptor cross-linking, Fas--Fas ligand interactions, and glucocorticoid treatment all depend on functional ALG-2, which is likely to be a part of the common pathway leading to cell death (Vito, P. et al., supra).
The ALG-2 sequence predicts an acidic protein of 191 amino acids with 2 EF-hand Ca.sup.2+ -binding domains; both EF-hand domains are required for Ca.sup.2+ binding. ALG-2 is the first Ca.sup.2+ -binding protein that has been shown to be directly required in the apoptosis pathway (Vito, P. et al., supra). A requirement for Ca.sup.2+ in apoptosis was previously suggested by studies showing its involvement in DNA cleavage (Hewish, D. R. and L. A. Burgoyne (1973) Biochem. Biophys. Res. Comm. 52:504-510). Other studies show that: 1) intracellular Ca.sup.2+ concentrations increase when apoptosis is triggered in thymocytes by either T cell receptor cross-linking or by glucocorticoids, and 2) cell death can be prevented by blocking this increase in intracellular Ca.sup.2+ (McConkey, D. J. et al. (1989) J. Immunol. 143:1801-1806; McConkey, D. J. et al. (1989) Arch. Biochem. Biophys. 269:365-370).
Additional support for the role of Ca.sup.2+ in apoptosis comes from work on Fas-mediated cell death (Vignaux, F. et al. (1995) J. Exp. Med. 181:781-786; Oshimi, Y. and S. Miyazaki (1995) J. Immunol. 154:599-609).
The discovery of polynucleotides encoding human apoptosis-related calcium-binding protein, and the molecules themselves, provide a means to investigate the regulation of programmed cell death and apoptosis. Discovery of molecules related to mouse ALG-2 satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the detection, prevention, and treatment of cancer, autoimmune diseases, lymphoproliferative disorders, psoriasis, atherosclerosis, restenosis, AIDS, immunodeficiency diseases, ischemic injuries, neurodegenerative diseases, osteoporosis, myelodysplastic syndromes, toxin-induced diseases, cachexia, and viral infections.